| 1. |
- Bergman, Inger, et al.
(författare)
-
The influence of sulphate deposition on the seasonal variation of peat pore water methyl Hg in a boreal mire
- 2012
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:9, s. e45547-
-
Tidskriftsartikel (refereegranskat)abstract
- In this paper we investigate the hypothesis that long-term sulphate (SO42-) deposition has made peatlands a larger source of methyl mercury (MeHg) to remote boreal lakes. This was done on experimental plots at a boreal, low sedge mire where the effect of long-term addition of SO42- on peat pore water MeHg concentrations was observed weekly throughout the snow-free portion of 1999. The additions of SO42- started in 1995. The seasonal mean of the pore water MeHg concentrations on the plots with 17 kg ha(-1) yr(-1) of sulphur (S) addition (1.3 +/- 0.08 ng L-1, SE; n=44) was significantly (p<0.0001) higher than the mean MeHg concentration on the plots with 3 kg ha(-1) yr(-1) of ambient S deposition (0.6 +/- 0.02 ng L-1, SE; n=44). The temporal variation in pore water MeHg concentrations during the snow free season was larger in the S-addition plots, with an amplitude of >2 ng L-1 compared to +/-0.5 ng L-1 in the ambient S deposition plots. The concentrations of pore water MeHg in the S-addition plots were positively correlated (r(2)=0.21; p=0.001) to the groundwater level, with the lowest concentrations of MeHg during the period with the lowest groundwater levels. The pore water MeHg concentrations were not correlated to total Hg, DOC concentration or pH. The results from this study indicate that the persistently higher pore water concentrations of MeHg in the S-addition plots are caused by the long-term additions of SO42- to the mire surface. Since these waters are an important source of runoff, the results support the hypothesis that SO42- deposition has increased the contribution of peatlands to MeHg in downstream aquatic systems. This would mean that the increased deposition of SO42- in acid rain has contributed to the modern increase in the MeHg burdens of remote lakes hydrologically connected to peatlands.
|
|
| 2. |
- Engström, Anna, et al.
(författare)
-
Detection of Rifampicin Resistance in Mycobacterium tuberculosis by Padlock Probes and Magnetic Nanobead- Based Readout
- 2013
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:4, s. e62015-
-
Tidskriftsartikel (refereegranskat)abstract
- Control of the global epidemic tuberculosis is severely hampered by the emergence of drug-resistant Mycobacterium tuberculosis strains. Molecular methods offer a more rapid means of characterizing resistant strains than phenotypic drug susceptibility testing. We have developed a molecular method for detection of rifampicin-resistant M. tuberculosis based on padlock probes and magnetic nanobeads. Padlockprobes were designed to target the most common mutations associated with rifampicinresistance in M. tuberculosis, i.e. at codons 516, 526 and 531 in the gene rpoB. Fordetection of the wild type sequence at all three codons simultaneously, a padlock probe and two gap-fill oligonucleotides were used in a novel assay configuration, requiring three ligation events for circularization. The assay also includes a probe for identificationof the M. tuberculosis complex. Circularized probes were amplified by rolling circle amplification. Amplification products were coupled to oligonucleotide-conjugatedmagnetic nanobeads and detected by measuring the frequency-dependent magneticresponse of the beads using a portable AC susceptometer.
|
|
| 3. |
- Göransson, Jenny, et al.
(författare)
-
Rapid Identification of Bio-Molecules Applied for Detection of Biosecurity Agents Using Rolling Circle Amplification
- 2012
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:2, s. e31068-
-
Tidskriftsartikel (refereegranskat)abstract
- Detection and identification of pathogens in environmental samples for biosecurity applications are challenging due to the strict requirements on specificity, sensitivity and time. We have developed a concept for quick, specific and sensitive pathogen identification in environmental samples. Target identification is realized by padlock- and proximity probing, and reacted probes are amplified by RCA (rolling-circle amplification). The individual RCA products are labeled by fluorescence and enumerated by an instrument, developed for sensitive and rapid digital analysis. The concept is demonstrated by identification of simili biowarfare agents for bacteria (Escherichia coli and Pantoea agglomerans) and spores (Bacillus atrophaeus) released in field.
|
|
| 4. |
- Ke, Rongqin, et al.
(författare)
-
Improving Precision of Proximity Ligation Assay by Amplified Single Molecule Detection
- 2013
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:7
-
Tidskriftsartikel (refereegranskat)abstract
- Proximity ligation assay (PLA) has been proven to be a robust protein detection method. The technique is characterized by high sensitivity and specificity, but the assay precision is probably limited by the PCR readout. To investigate this potential limitation and to improve precision, we developed a digital proximity ligation assay for protein measurement in fluids based on amplified single molecule detection. The assay showed significant improvements in precision, and thereby also detection sensitivity, over the conventional real-time PCR readout.
|
|
| 5. |
- Mathot, Lucy, et al.
(författare)
-
Automated Genotyping of Biobank Samples by Multiplex Amplification of Insertion/Deletion Polymorphisms
- 2012
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:12
-
Tidskriftsartikel (refereegranskat)abstract
- The genomic revolution in oncology will entail mutational analyses of vast numbers of patient-matched tumor and normal tissue samples. This has meant an increased risk of patient sample mix up due to manual handling. Therefore, scalable genotyping and sample identification procedures are essential to pathology biobanks. We have developed an efficient alternative to traditional genotyping methods suited for automated analysis. By targeting 53 prevalent deletions and insertions found in human populations with fluorescent multiplex ligation dependent genome amplification, followed by separation in a capillary sequencer, a peak spectrum is obtained that can be automatically analyzed. 24 tumor-normal patient samples were successfully matched using this method. The potential use of the developed assay for forensic applications is discussed.
|
|
| 6. |
- Mezger, Anja, et al.
(författare)
-
Detection of Rotavirus Using Padlock Probes and Rolling Circle Amplification
- 2014
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:11, s. e111874-
-
Tidskriftsartikel (refereegranskat)abstract
- Rotavirus infections are one of the most common reasons for hospitalizations due to gastrointestinal diseases. Rotavirus is often diagnosed by latex agglutination assay, chromatography immunoassay, or by electron microscopy, which are all quite insensitive. Reverse transcription polymerase chain reaction, on the other hand, is very sensitive to variations at the genomic level. We developed a novel assay based on a set of 58 different padlock probes with a detection limit of 1,000 copies. Twenty-two patient samples were analyzed and the assay showed high concordance with a PCR-based assay. In summary, we present a new assay for sensitive and variation tolerant detection of rotavirus.
|
|
| 7. |
- Moens, Lotte N., et al.
(författare)
-
Diagnostics of Primary Immunodeficiency Diseases : A Sequencing Capture Approach
- 2014
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:12, s. e114901-
-
Tidskriftsartikel (refereegranskat)abstract
- Primary Immunodeficiencies (PID) are genetically inherited disorders characterized by defects of the immune system, leading to increased susceptibility to infection. Due to the variety of clinical symptoms and the complexity of current diagnostic procedures, accurate diagnosis of PID is often difficult in daily clinical practice. Thanks to the advent of next generation'' sequencing technologies and target enrichment methods, the development of multiplex diagnostic assays is now possible. In this study, we applied a selector-based target enrichment assay to detect disease-causing mutations in 179 known PID genes. The usefulness of this assay for molecular diagnosis of PID was investigated by sequencing DNA from 33 patients, 18 of which had at least one known causal mutation at the onset of the experiment. We were able to identify the disease causing mutations in 60% of the investigated patients, indicating that the majority of PID cases could be resolved using a targeted sequencing approach. Causal mutations identified in the unknown patient samples were located in STAT3, IGLL1, RNF168 and PGM3. Based on our results, we propose a stepwise approach for PID diagnostics, involving targeted resequencing, followed by whole transcriptome and/or whole genome sequencing if causative variants are not found in the targeted exons.
|
|
| 8. |
- Raap, A. K., et al.
(författare)
-
Non-Random mtDNA Segregation Patterns Indicate a Metastable Heteroplasmic Segregation Unit in m.3243A>G Cybrid Cells
- 2012
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:12, s. e52080-
-
Tidskriftsartikel (refereegranskat)abstract
- Many pathogenic mitochondrial DNA mutations are heteroplasmic, with a mixture of mutated and wild-type mtDNA present within individual cells. The severity and extent of the clinical phenotype is largely due to the distribution of mutated molecules between cells in different tissues, but mechanisms underpinning segregation are not fully understood. To facilitate mtDNA segregation studies we developed assays that measure m.3243A>G point mutation loads directly in hundreds of individual cells to determine the mechanisms of segregation over time. In the first study of this size, we observed a number of discrete shifts in cellular heteroplasmy between periods of stable heteroplasmy. The observed patterns could not be parsimoniously explained by random mitotic drift of individual mtDNAs. Instead, a genetically metastable, heteroplasmic mtDNA segregation unit provides the likely explanation, where stable heteroplasmy is maintained through the faithful replication of segregating units with a fixed wild-type/m.3243A>G mutant ratio, and shifts occur through the temporary disruption and re-organization of the segregation units. While the nature of the physical equivalent of the segregation unit remains uncertain, the factors regulating its organization are of major importance for the pathogenesis of mtDNA diseases.
|
|
| 9. |
- Sun, Song, et al.
(författare)
-
Genome-Wide Detection of Spontaneous Chromosomal Rearrangements in Bacteria
- 2012
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:8, s. e42639-
-
Tidskriftsartikel (refereegranskat)abstract
- Genome rearrangements have important effects on bacterial phenotypes and influence the evolution of bacterial genomes. Conventional strategies for characterizing rearrangements in bacterial genomes rely on comparisons of sequenced genomes from related species. However, the spectra of spontaneous rearrangements in supposedly homogenous and clonal bacterial populations are still poorly characterized. Here we used 454 pyrosequencing technology and a 'split mapping' computational method to identify unique junction sequences caused by spontaneous genome rearrangements in chemostat cultures of Salmonella enterica Var. Typhimurium LT2. We confirmed 22 unique junction sequences with a junction microhomology more than 10 bp and this led to an estimation of 51 true junction sequences, of which 28, 12 and 11 were likely to be formed by deletion, duplication and inversion events, respectively. All experimentally confirmed rearrangements had short inverted (inversions) or direct (deletions and duplications) homologous repeat sequences at the endpoints. This study demonstrates the feasibility of genome wide characterization of spontaneous genome rearrangements in bacteria and the very high steady-state frequency (20-40%) of rearrangements in bacterial populations.
|
|
| 10. |
- Weibrecht, Irene, et al.
(författare)
-
Simultaneous visualization of both signaling cascade activity and end-point gene expression in single cells
- 2011
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 6:5, s. e20148-
-
Tidskriftsartikel (refereegranskat)abstract
- We have developed an approach for simultaneous detection of individual endogenous protein modifications and mRNA molecules in single cells in situ. For this purpose we combined two methods previously developed in our lab: in situ proximity ligation assay for the detection of individual protein interactions and -modifications and in situ detection of single mRNA molecules using padlock probes. As proof-of-principle, we demonstrated the utility of the method for simultaneous detection of phosphorylated PDGFRβ and DUSP6/MKP-3 mRNA molecules in individual human fibroblasts upon PDGF-BB stimulation. Further we applied drugs disrupting the PDGFRβ signaling pathway at various sites to show that this combined method can concurrently monitor the molecular effect of the drugs, i.e. inhibition of downstream signaling from the targeted node in the signaling pathway. Due to its ability to detect different types of molecules in single cells in situ the method presented here can contribute to a deeper understanding of cell-to-cell variations and can be applied to e.g. pinpoint effector sites of drugs in a signaling pathway.
|
|
